Grace W.-J. Lin

Effect of ethanol feeding during pregnancy on placental transfer of alpha-aminoisobutyric acid in the rat

Abstract The influence of ethanol consumption during pregnancy on maternal-fetal transfer of amino acid was studied. Pregnant rats were fed a liquid diet containing 30% ethanol-derived calories from gestation-day 6 to 21; control rats were pair-fed identical diets, except that sucrose substituted isocalorically for ethanol. On gestation-day 21, 2 uCi/100 g body weight of 14C-alpha-aminoisobutyric acid (14C-AIB) was injected into the maternal circulation, and 90 minutes later maternal blood and liver, placentas and fetuses were removed for radioactivity measurement. No differences between ethanol-fed and control rats in the distribution of 14C-AIB in maternal plasma or the uptake of 14C-AIB by the maternal liver were observed. However, the radioactivities in placenta and fetal tissues suffered a significant 20 to 40% reduction in the ethanol-fed group, suggesting that ethanol feeding during pregnancy impairs placental function.

Significance of the Gastrointestinal Tract in the in Vivo Metabolism of Ethanol in the Rat

The rate of ethanol metabolism and the extrapolated zero time blood ethanol concentration (C0) were compared in naive and ethanol-fed rats following intracardiac or ingastric administration of a test dose of ethanol (3 g/kg). If the gastrointestinal tract is involved in the disposition of ethanol, intragastric administration should result in a lower C0 and a faster overall rate of metabolism than intracardiac administration, since part of the dose would be metabolized in the gastrointestinal tract without having been absorbed and thereby entering the blood. However, no significant differences were observed in C0. The rate of metabolism was substantially higher in the ethanol-fed rats, but was uninfluenced by route of administration. Thus, the gastrointestinal tract plays no significant role in the metabolism of ethanol.

Maternal-fetal folate transfer: effect of ethanol and dietary folate deficiency

The effects of acute ethanol treatment and dietary folate deficiency on maternal-fetal folate transfer were studied to asses the hypothesis that the potentiation of ethanols toxic effect on the fetus during ongoing folate deficiency was due to the impairment of folate transfer. Sprague-Dawley rats were fed either a folate-deficient diet (0.2 mg/kg) or a folate-sufficient diet (2 mg/kg) for an average of 11 weeks before pregnancy and continued until gestation day 11 when they were sacrificed. On gestation day 11, pregnant rats were treated with either ethanol (2.5 g/kg body weight) or isocaloric sucrose (control) followed by an intravenous administration of 3H-folate (2 muCi/100 g body weight) at 120 minutes. At 210 minutes, maternal blood and feto-placental tissues were removed for radioactivity measurement. Folate status and ethanol treatment had no effect on the distribution of 3H-folate in maternal circulation. However, contrary to the hypothesis, the uptake of 3H-folate by feto-placental tissues was increased in folate deficiency and by ethanol treatment, indicating that folate transfer was not impaired by the ethanol treatment. Other possibilities are discussed.

Effect of ethanol consumption during pregnancy on folate coenzyme distribution in fetal, maternal, and placental tissues

Abstract Ethanol has been shown to be a teratogen in humans and experimental animals; however, the mechanism of its actions is not known. Because ethanol interferes with the metabolism of folate coenzymes that participate in various biosynthetic pathways of nucleic acids, the effect of gestational ethanol consumption on the distribution of folate coenzymes in tissues was investigated with an eye to the possibility that such effects might shed light on the mechanism by which ethanol produces its teratogenic effect. Sprague-Dawley rats were fed a 35% ethanol-calorie liquid diet from gestation days 7–21; controls were pair-fed with isocaloric sucrose substituted for ethanol. Rats were killed on day 21. Food intake, maternal weight gain, litter size, and conceptus weight were similar between ethanol and control rats. However, fetal weight was decreased and placental weight increased in ethanol group as compared with the control. Total folates and folate coenzymes were determined in fetal liver and brain, placenta, and maternal liver. Ethanol consumption decreased 5-methyltetrahydrofolate in the placenta, which was compensated by increasing tetrahydrofolate and formyltetrahydrofolates. In fetal liver, formyltetrahydrofolates and formiminotetrahydrofolate were decreased and tetrahydrofolate increased with ethanol. In fetal brain, ethanol not only decreased total folates but also altered the coenzyme pattern by decreasing 5-methyltetrahydrofolate and increasing tetrahydrofolate and formyltetrahydrofolates. These changes in folate coenzyme pattern indicate ethanol-induced alteration in folate metabolism. The possible physiologic significance of these changes in relation to Fetal Alcohol Syndrome is discussed.

Effect of dietary folic acid levels and gestational ethanol consumption on tissue folate contents and rat fetal development

Abstract It has been shown that folate deficiency potentiates the teratogenicity/embryotoxicity of ethanol. In this study the degree of folate deficiency in exacerbating ethanols teratogenic/embryotoxic potential was assessed. Four levels of folate were tested. Female rats were deprived of folate for three weeks to lower their folate stores. They were then divided into four groups and fed diets containing 0.05, 0.20, 0.50 or 2.00 mg folic acid/kg diet (1% succinylsulfathiazole). After two weeks they were mated and were continued on their respective diets during pregnancy. On day 11, ethanol (5 g/kg body weight in two divided doses four hrs apart) was administered to half of the rats in each group intragastrically. The other half of the rats that served as controls were given isocaloric sucrose in the same manner. Food intakes were similar in all groups. Rats were killed on day 21. Folate levels were determined in six tissues: maternal liver and plasma, placenta, fetal liver, brain and plasma. Folate deficient, ethanol-treated groups produced less viable fetuses that were correlated with tissue folate levels (L. casei procedure), which, in turn, correlated with folate levels in the diets. There is a relationship between the degree of folate deficiency and the ethanols adverse effect: the more severe the folate deficiency the more potent the teratogenic/embryotoxic effect of ethanol. The numbers of dead and resorbed fetuses expressed as a percentage of total implants were (ethanol vs control): 0.05 mg folate/kg diet, 38.9 vs 18.9; 0.20 mg/kg, 23.1 vs 6.8; 0.50 mg/kg, 16.7 vs 8.4. But when rats were fed sufficient folic acid (2 mg/kg diet), ethanol treatment had no effect on fetal outcome. These observations strongly suggest that maternal folate deficiency may play an important role in the fetal alcohol syndrome.

METABOLISM OF PYRAZOLE IN THE RAT1

Hydrolysis and chromatography of urine from male Sprague-Dawley rats injected with pyrazole-3,4−14C (PC14), and comparison with authentic material, have shown that the major metabolite of pyrazole (P) in rats is 4-hydroxypyrazole, excreted in hydrolyzable conjugated forms. Urinary unchanged P is usually less than 10% of the injected dose. The distribution of PC14 in 20 tissues 4 hours after IP injection of 1 mmole/kg PC14 showed low levels in fat (0.4 mM) and skin (0.9 mM) and high levels in stomach (4.4 mM); other tissues had levels of about 1 mM. Kinetics of Ps disappearance from blood are complicated; the curves (after IP doses of 0.2, 1.0, 2.0, and 4.0 mmoles/kg) are sigmoidal and may indicate induction of a hydroxylating enzyme. When an IP dose of 2.0 mmoles/kg PC14 was given at various blood levels of ethanol (maintained by infusion), not only was alcohol metabolism greatly slowed, but P oxidation was essentially stopped at high (300 mg/100 ml) blood levels of ethanol; under these circumstances, the percentage of unchanged urinary P rose to 75%.

Folate deficiency and acute ethanol treatment on pregnancy outcome in the rat

Abstract The interaction of folate deficiency and acute ethanol (EtOH) treatment on tissue folate level and fetal outcome was investigated. Sprague-Dawley rats (50-d-old) were fed folate deficient (no folic acid added) or folate sufficient (2 mg folic acid/kg) diet containing 1% succinylsulfathiazole for 7 weeks prior to and during gestation. On day 11 EtOH was administered intragastrically to half of the deficient and sufficient rats (5g/kg in 2 divided doses 4 hrs apart). Other halves served as controls were given isocaloric sucrose in the same manner. Rats were killed on day 21; pregnancy outcome and tissue folate levels were examined. Total folate values in maternal plasma and liver and placenta as well as in fetal liver and brain were all reduced in deficient groups. EtOH treatment had no effect on tissue folate level. Although food intakes were not different among groups throughout the experiment, maternal weight gains during gestation differed. Deficient groups gained much less, had more dead and resorbed fetuses and smaller live fetuses. Between the two deficient groups, EtOH treated rats produced three times more dead and resorbed fetuses than folate deficient alone. On the contrary, in folate sufficient groups EtOH had no effect on pregnancy outcome. It is concluded that under poor folate nutrition even a moderate EtOH given at a critical period of fetal development can severely impair fetal development.

In vitro inhibition of catalase by 4-hydroxypyrazole, a metabolite of pyrazole

Pyrazole is an effective inhibitor of catalase (EC 1.11.1.6) when given in vivo, but not in vitro [1,2]. It has been suggested [3] that the in vivo inhibition is mediated by ‘a product from the metabolism of pyrazole by the microsomal mixedfunction oxidase system. . .in a process which can be prevented by alcohol.’ From a study of the action of pyrazole on tryptophan oxygenase (EC 1.13.1_12), another hemoprotein enzyme, it has also been proposed that the delayed in vivo inhibition seen after pyrazole treatment, much stronger than any comparable in vitro inhibition, results from either an inhibitory action on tryptophan oxygenase synthesis and/or a direct interference with the heme prosthetic group by an active derivative of pyrazole [4]. Work in our laboratory [S] has revealed that the major metabolite of pyrazole in rats is 4-hydroxypyrazole, excreted in hydrolyzable conjugated forms. Production of this metabolite is inhibited by ethanol. We now demonstrate that 4-hydroxypyrazole is a good inhibitor of catalase in vitro.

Effect of ethanol feeding on the urinary histamine level of the pregnant rat.

The influence of ethanol feeding during pregnancy on histamine excretion was studied. Pregnant Sprague-Dawley rats (N = 5) were fed a liquid diet containing 30% ethanol-derived calories from gestation-day 7 to 21; control rats (N = 5) were pair-fed with isocaloric sucrose substituted for ethanol. Twenty-four hour urines were collected for histamine analysis. Rats were killed on day 21 of gestation. Food and ethanol intakes averaged 260 kcal and 11 g/kg/day, respectively. No differences were found between ethanol and control rats in maternal weight gain, litter size or in fetal and placental weights. Although urinary histamine increased in all rats with the advance of pregnancy, on day 16, ethanol rats excreted significantly more (47%) than the controls (199.1 +/- 33.9 vs 135.5 +/- 51.4 ug/24 hr); on day 20, it was 123% more (534.6 +/- 114.4 vs 239.5 +/- 99.3 ug/24 hr). Ethanol enhanced urinary histamine did not reflect the histamine content or histidine decarboxylase activity of fetal liver, presumed site of histamine formation; its physiological significance is discussed.

Distribution of Ethanol between Blood and Dorsal Subcutaneous Air of the Rat and the Feasibility of Using this Distribution Coefficient to Estimate Blood Ethanol

Equilibration of ethanol between dorsal subcutaneous air and blood is rapid, a steady state being already present in 60 seconds after air injection in the rat. The mean in vivo partition coefficient (blood/air) is 2,875, the linear regression coefficient having a standard error of 112, the high F value of this regression indicating the linearity and reliability of the estimates of calculated blood ethanol; the variance arises mainly from temperature variance of the air bleb. Although the concentration of alcohol in the blood must remain the standard of comparison, the production of the air bleb and its subsequent serial sampling is a virtually noninvasive technique which yields an almost immediate result at a sensitivity and specificity greater than that from the usual enzymatic determination of ethanol in blood.